Multigene phylogeny and morphological descriptions of five species of Agaricus sect. Minores from subtropical climate zones of Pakistan

The genus Agaricus includes more than 500 species mostly containing the edible and cultivated species worldwide. As part of the ongoing studies on the biodiversity of genus Agaricus in Pakistan, our objective was to focus on A. sect. Minores which is the largest section of the genus. In the first phylogenetic analyses based on the ITS region of the nuclear ribosomal DNA, our sample included specimens of 97 named species, 27 unnamed species, and 31 specimens (29 newly generated sequences in this study) from subtropical climate zones of Pakistan that likely belong to this section based on their morphology. The 31 specimens grouped into five distinct, well-supported clades corresponding to five species: A. glabriusculus already known from Pakistan and India, A. robustulus first recorded from Pakistan and briefly described here but already known from Bénin, Malaysia, China, and Thailand, and three possibly endemic new species described in detail A. badiosquamulosus sp. nov., A. dunensis sp. nov., and A. violaceopunctatus sp. nov. The sixth species currently known in Pakistan, including A. latiumbonatus also found in Thailand, were included in a multigene tree based on ITS, LSU, and Tef-1α sequence data. They all belong to a large pantropical paraphyletic group while most temperate species belong to a distinct clade, which includes about half of the species of the section. The current study aims to propose three novel species of genus Agaricus based on comprehensive morphological as well as molecular phylogenetic evidences from Pakistan.


Introduction
The genus Agaricus (Basidiomycota) is the type genus of Agaricaceae containing saprophytic species.The species are adapted to live in various climatic regions as subtropical, tropical, temperate, Caribbean islands and deserts.Several species are consumed and cultivated because of their nutritional and medicinal importance such as A. bisporus (J.E.Lange) Imbach, A. arvensis Schaeff.and A. subrufescens Peck [1, [2][3][4].
The classification of the genus is mainly proposed by Zhao et al. [5] which was revised using molecular clock methods [6].Five monophyletic clades A, B, C, D, and E were proposed having stem ages ranging from 30-33 My ago.Each clade is considered as subgenus.Amongst these five subgenera, Clade A is considered as a subgenus Minores [6].It includes mainly the section Minores reported previously by Zhao et al. [5] that diverged 30 My ago and is subdivided into Clade A1/ sect.Minores, Clade A2/sect.unknown or sect.Laeticolores and sect.Laeticolores.Agaricus sect.Laeticolores was represented by only one species A. rufoaurantiacus (LAPAM15) [6] which was reported as subgen.Minoriopsis by Chen et al. [7].In the same study by Chen et al. [7] another new section Leucocarpi has been introduced and Clade A2/ sect. 1 still remains unnamed.The classification of subgen.Minores is recently revised where subgen.Minoriopsis is splitted into A. sect.Kerrigania and Minoriopsis.Agaricus sect.Minores is sub-divided into three sections viz. A. sect.Pantropicales L.A. Parra, Angelini, B. Ortiz, Linda J. Chen & Callac, A. sect.Minores (Fr.) Henn., and A. sect.Leucocarpi Linda J. Chen & Callac [6][7][8].Later, He et al. [9] has introduced a new section of Agaricus subg.Pseudochitonia and now the number of sections are increased to 24.According to Index Fungorum data, there are about 6000 species of Agricus recorded from all over the world so far (accessed in April 2022).
Generally the species of A. sect.Minores are distributed world wide in tropical and temperate areas [5,10] and some are found in extreme climates like secotioid species, adapted well to desert conditions [11,12].However, the newly reported species in this study were collected mainly from subtropical areas of Pakistan and the desert but none of them is sectioned.Previously 35 species of the genus Agaricus have been reported from Pakistan [7,[13][14][15][16][17][18][19][20] amongst these only two species belong to Agaricus sect.Minores [17].Recently, two new species viz. A. midnapurensis and A. purpureosquamulosus and a new record of A. glabriusculus have been documented using ITS region from India [21] and Agaricus parviumbrus is documented as a new species in Fungal Planet description sheets [22].
The data of all the species in this study is a significant contribution to the knowledge of genus Agaricus from Pakistan.A few studies have been conducted based on multigene and morphological data so far.The heteromorphic sites of ITS region within and between the closely related taxa are analyzed in this study.

Specimen collection and sampling sites description
A total of 29 specimens of genus Agaricus were collected from different semi-arid and arid localities of Punjab province, Pakistan.Based on morphological observations and fresh basidiomata field notes, the specimens likely belonged to Agaricus sect.Minores.As part of the current study on the genus, surveys were conducted in different subtropical areas of Punjab province i.e., Lahore; Toba Tek Singh; Haroonabad, district Bahawalnagar and Lal Suhanra National Park, Cholistan desert, district Bahawalpur, Pakistan.
Lal Suhanra National Park lies in southeastern Punjab, Pakistan.This national park is part of the Cholistan desert having fungal flora that has not yet been studied so far.It is a subtropical region characterized by very low annual rainfall (90-200 mm) in monsoon season, low relative humidity (60%) and high temperature (50˚C) in summer [23].It is located 32 km away from district Bahawalpur.District Bahawalpur lies in southern side of Punjab, Pakistan.This subtropical region receives average annual rainfall fluctuating from 90 to 200 mm only [24].The average annual temperature is more or less similar to Lal Suhanra National Park ranging from 6 to 10˚C in winters and 49 to 52˚C in summers [25].
The other collection site lies in Haroonabad, District Bahawalnagar, Punjab, Pakistan adjacent to Cholistan desert which is characterized by hot and dry climate with an extreme temperature of 50˚C and minimum temperature of 11˚C.The average annual rainfall is just 99 mm [26,27].The basidiomata (BWN 34, BWN 67, BWN 85) were collected along the bank of three R (3R) irrigation canal under Acacia nilotica from the loamy soil.
Toba Tek Singh is a district of Punjab with a subtropical climate.The maximum temperature is recorded at 40.7˚C and a minimum of 6˚C with an average annual rainfall of 254-381 mm [28].
Changa Manga Forest lies in the southeast of the district Lahore.This is a subtropical area with minimum to maximum temperature ranges from 5.9 to 39.6˚C, respectively, and the annual rainfall is about 650 mm [29].

Morphological characterization
The fresh specimens were photographed during field surveys at collection sites.Collected basidiomata were carefully tagged and dried in sunlight or by using a fan heater.All the dried specimens were packed in zipper bags and brought to the laboratory for further processing.Morphological observations such as color, size, and shape of pileus, stipe, and lamellae were noted [30].Other fresh sporocarp characters were also observed and noted during collection survey including discoloration upon bruising/handling, odor, and different chemical reactions such as KOH and Schaeffer's reaction [31].Color codes were given from Munsell's Soil Color Charts [32].
Tissues from different parts of fruiting bodies were rehydrated in 5% KOH (w/v) solution followed by staining in 1% aqueous Congo red (w/v).Anatomical characeterization included size and shape of basidia, basidiospores, cheilocystidia, pileipellis and stipitipellis and their measurements were taken using a light microscope at 100X (MX4300H, Meiji Techo Co., Ltd., Japan).All the microscopic features were represented by at least 20 measurements each.The dimensions of basidiospores are provided as (a) b-c (d) × (e) f-g (h), [avX, Qm, n = i × j] where b-c and, f-g includes the spore length and width, respectively, between the 5th percentile and the 95th percentile, (a) and (d) the minimum or maximum values of spore lengths recorded, similarly (e) and (h) the minimum or maximum values of basidiospores width were recorded, avX the mean of length by width ± SD (standard deviation), Qm the mean of Q coefficient (length/width ratio), n represents the total number (i) of basidiospores measurements of each sample/collection and (j) indicates number of total samples/collections measured.Measurements of other anatomical structures as basidia, cheilocystidia, hyphal structures including pileipellis, and stipitipellis were available in dried samples included the range between the minimum or maximum values calculated in length and width.The measurement of basidia was considered excluding sterigmata.Short descriptions of all the new species have been deposited in MycoBank.
DNA extraction, PCR amplification and DNA sequencing.DNA was extracted from the dried specimens using the 2% CTAB method with some modifications as described by Zhao et al. 2011 [5].PCR amplification was performed of three nrDNA regions viz.ITS (Internal Transcribed Spacer region) using ITS1F/ITS4 primers [33,34] LSU (large subunit fragment) using LROR/LR5 set of primers [35] and EF1 (translation elongation factor) utilizing EF1-983F/EF1-1567R combination of primers [36].In PCR cycle, denaturation was done at 95˚C followed by annealing of 35 cycles at 94˚C and final extension at 72˚C.The PCR products were sequenced from BGI (Beijing Genomic Institute) and Genewiz, UK.All the newly generated sequences during this study have been submitted to GenBank.
DNA sequence alignments.Sequence alignment for ITS and multigene datasets was performed independently, using T-cofee version 8.99 [37] then final adjustment was done manually in Bioedit version 7.2.0.The CIPRES PORTAL v. 3.1 [38] was used for maximum likelihood (ML) analyses.Both ITS and multigene phylogeentic analyses were conducted for each alignment separately using RAXML-HPC2 v 8.1 [39].Topology of phylogram was retrieved at 1000 bootstrap replicates.Significant support was considered to be BS�50.No significant inconsistency between the ITS and multigene sequence data was observed, therefore ITS, LSU, and TEF1 sequences were integrated in BioEdit for final phylogenetic analyses.The multigene data was segmented into ITS, LSU, TEF1 (intron and coding sites).
Data analyses.The most suitable substitution model for each region was inferred with MrModeltest version 2.2 [40]: SYM+G for all partitioned DNA regions.Bayesian Inference (BI) [41] was completed with MrBayes v. 3.1.2.Six Markov chains were run for one million generations and sampled every 100th generation.Burn-in was indicated in TRACER v. 1.6 [41] from trace plots of likelihood and finally discarded.The resulting phylograms were opened in FigTree version 1.4.2(http://tree.bio.ed.ac.uk/sofware/fgtree/).The final alignments of both ITS and multigene dataset were deposited in TreeBASE (ID = 30723).
Species-specific ITS markers.Taxa, clades, sub-sections, or sections characterizing markers were located in the final alignment of 153 total ITS sequences of A. sect.Minores.These markers are provided with capital letters and are shown along with flanking sequences.IUPAC codes, for example Y, do not indicate ambiguous nucleotide (C or T) but heteromorphic site (C and T) likely reflecting allelic polymorphic conditions in the heterokaryotic (n+n) basidiomata.Deletions or insertions are mentioned in square brackets.The locality/position of the markers are numbered with respect to their position either in the 5'-3' ITS1-5.8S-ITS2sequence data (starting from tygaatt) of the taxon to be characterized or in the final alignment deposited in TreeBASE for the clades or higher taxa characterization.

Phylogenetic analyses
The phylogenetic analyses comprised of 42 sequences that were newly generated (30 ITS, 06 LSU, and 06 TEF1) from 30 specimens.These 30 specimens belonged to six species including five new species described in this study and a new record (A. robustulus) from Pakistan.ITS sequences of all the specimens were included for the first time in phylogenetic analyses.The alignment for ITS dataset consisted of total sequence of 158 specimens and 718 characters (Fig 1).Among these, seven samples were used as outgroup belonging to seven different species of: A. sect.Leucocarpi (A. leucocarpus), A. subg.Minoriopsis (A. rufoaurantiacus and A. sp.ZRLWXH3064), A. sect.Pantropicales (A. candidolutescens, A. sp.LAPAM14 and A. sp.ZRLWXH3161) and A. sect.Agaricus (A. campestris).All samples with their GenBank accession numbers are listed in Table 1 and references in Table 2.
The alignment for multi-gene analyses consisted of sequences (ITS/LSU/TEF1) of 105 sequences and 2052 characters (Fig 2).The five samples used as outgroup belonging to five different species of: A. sect.Leucocarpi (A. leucocarpus), A. sect.Pantropicales (A. candidolutescens, A. sp.LAPAM14 and A. sp.ZRLWXH3161) and A. sect.Agaricus (A. campestris).Agaricus huijsmanii, a European species, is separated from all the clades in multigene phylogenetic analyses as referenced in He et al. 2017 [32] also, however, this taxon got aligned within A. sect.Minores in ITS phylogenetic analyses.The remaining 99 samples belonged to 96 species or putative species of A. sect.Minores (Table 1).
For both multigene and ITS analyses, the topologies of the phylogenetic trees using the Maximum likelihood (ML) or the Bayesian inference (BI) methods were highly similar.The bootstrap support values (BS) and Bayesian posterior probabilities (PP) are reported in the ML phylogenetic trees of Fig 1 for ITS analyses, and Fig 2 for multigene analyses.The multigene data of all the newly reported species and those available of previously reported samples were completed in our multigene analyses, however, still there remain many species that only have ITS sequence data.For this reason, we presented first the phylogeny of the sections based on multigene-analyses.The ITS analyses remain useful mainly at the species level to delimit new taxa and identify their most closely related taxa.
Diagnosis: This species is characterized by having a dark brown pileus covering with purplish tinged squamules, immediately gives strong fulvescent discoloration when bruised.Annulus membranous, single edged with erroded edges in most of specimens and superous.Basidia frequently bisporic to tetrasporic and monosporic observed in only one collection, cheilocystidia abundant, multi-septate and broadly clavate.
Agaricus dunensis H. Bashir & M. Asif (Figs 5-7) Heteromorphisms are shown by yellow highlight.Newly added samples are indicated in bold. https://doi.org/10.1371/journal.pone.0302222.t003 Etymology: 'dunensis' in Latin referred to sand dune area from where the type specimen was recorded for the first time.
Diagnosis: This species is distinguished well having a pileus with bright yellow to rust squamules becoming brownish at maturity, pileus and stipe surface immediately turning dark brown when touched, and a strong red to blackish-brown discoloration upon bruising, monosporic basidia frequently observed in one sample (L2), frequently bisporic to tetrasporic in others, cheilocystidia polymorphous.
Description: Pileus 2.5-9 cm in diam., convex then plano-convex, slightly appressed and flattened at disc (LS4), sometimes disc depressed (BWN-64) at maturity, thoroughly covered by orange-yellow (5.8YR 5.9/6.2) squamules (predominant in L99 collection) in young stages, at maturity surface has dotted bright yellow (2.1Y 4.1/5.4)squamules becoming yellowish brown (10YR6/8) in some fruiting bodies, few scattered towards margin more dense at the center in mature sporocarp on a creamy (3.1G 8.5/0.7)background.Surface dull and dry.Margins slightly appendiculate, white, slightly incurved when young become straight at maturity, slightly exceeding lamellae.Lamellae light pink (7.2R 7.5/2) at young stage to dark brown (5.7R 2.2/1.4) at maturity, free, crowded, intercalated with lamellulae, entire edges sometimes slightly wavy.Stipe 1.5-6.5 × 0.5-3 cm, cylindrical to slightly bulbous at the base to ventricose with bulbous from center tapering towards base (BWN-64), stuffed, provided with an annulus in its upper part close to lamellae, covered with creamy white squamules from above and below annulus, creamy when young becomes brown from top at maturity, discoloring yellow or rusty at stipe base when rubbed, becoming light yellowish brown (1.3Y 6/2.4) when cut in L2, no significant discoloration observed in L99, brown (9.7YR 5.5/2.8)color appears mildly at stipe base in other specimens.Annulus superous, single edged, white (8.2PB 9.3/0.9),ruptured at young stage leaving behind a ring zone at maturity, fragile, smooth, membranous or thick in contrast, A. dunensis gave no significant discoloration when cut or very light yellow observed (L2) or faint yellowish brown at stipe base (BWN34 and BWN64).Agaricus parvibicolor has ellipsoid to elongate basidiospores having an average size of 6.3 × 3.9 μm [31] while A. dunensis has subglubose to ellipsoid basidiospres with an average size of 6.5 × 5. Material examined: PAKISTAN, Punjab, University of the Punjab, Lahore, at 217 m a.s.l., solitary or in groups on rich loamy soil on the grassy grounds of the University Botanical Garden, 13 July 2016, Hira Bashir, PU217 (LAH35764).

Notes:
Phylogenetically, four of our specimens get clustered with a recently described Chinese taxon, A. robustulus.This species is characterized by its reddish brown squamules on pileus and strongly fulvescent discoloration in mature sporocarps when rubbed or bruised.Cheilocystidia are polymorphic, having simple, broadly clavate to pyriform shaped.All these morphological characters are similar to that reported by Chen et al. 2017 [7].Other species of this section also resemble morphologically to A. brunneolus, A. goossensiae, and A. megalosporus.However, the spore size in A. goosensiae is bigger (6.3 × 4.4) and cheilocystidia are inconspicuous while the other two taxa can be distinguished having large sized pileus exceeding 7 cm in diameter.This species is first time recorded from Pakistan.

Agaricus glabriusculus S. Hussain (Figs 8D-8F and 10)
Diagnosis: This species is distinguished well by having a pileus with brown fibrillose squamules dense at disc, context with pale brown discoloration when cut, annulus not very conspicuous, fragile, non-persistent and left in the form of ring zone on upper half of stipe.Basidia frequently bisporic to tetrasporic, cheilocysytidia broadly clavate and some have globose elements at apices.
Habit, habitat and distribution: In groups, on grassy grounds of Botanical Garden, University of the Punjab, near Eucalyptus trees.This species is known only from Lahore, Pakistan until now.

Notes:
In phylogenetic analysis A. violaceopunctatus formed a subclade with all the undescribed taxa except A. microviolaceous.One of the closest taxon is the Pakistani specimen (MCR59), which was misidentified as A. goossensiae and the sequence is also submitted in GenBank (LAH5972011) with wrong identification.Our species has three nucleotide differences from A. sp.(MCR59) at position 37, 43 and 690 in ITS sequence data.When the original description of MCR59 (taken from the dissertation of author) was compared with our taxon, morphologically, pileus in MCR59 was recorded obtuse to umblicate with dark reddish squamules against reddish grey to hazel background and uplifted margin while in our species the squamules are strongly violaceous with creamy background.Annulus was not observed in MCR59 while in our collections annulus is very prominent, membranous and white.Clavate cheilocystidia were observed in MCR59 which are absent in our specimens.On the otherhand, A. microviolaceus also has violet squamules, small sized sporocaps as observed in our newly described taxon but phylogenetically both species are separated having more than 15 nucleotide differences in their ITS sequence data.So, our new species does not similar to any of the already described taxa.

Discussions
From Pakistan, three new species viz., A. badiosquamulosus, A. dunensis, and A. violaceopunctatus have been recorded during this study that are grouped within A. sect.Minores.In addition, one new record, A. robustulus, and an already reported species, A. glabriusculus, have also been documented providing their ITS, LSU, and Tef-1α sequence data.Two of those, A. badiosquamulosus and A. glabriusculus, clustered close to each other and are sister to A. sp.(MATA774) and with A. goossensiae (GF929; type specimen).Agaricus sp.(MATA774) was collected from a dune in 2007 growing near Tulostoma sp. by Philippe Callac & Ge ´rardo Mata 2 Km away from Montepio, San Andre ´s Tuxtle, Mexico.Agaricus cf.goossensiae (ADK2751) was initially used in the phylogenetic analysis by Zhao et al. 2011 [5] and later as A. sp.[7,9] so the species status was unclear.Therefore, in this study, we have requested to get a sequence of the type specimen of ADK2751 to Belgium herbarium which was provided to us for analysis.After comparisons, it was concluded that the ITS sequence of A. goossensiae (GF929) is far from the sequence of ADK2751 and both of these sequences do not share any genetic character.Therefore, ADK2751 is considered to be another taxon, different from A. goossensiae.So, the later concept was taken into account and accepted.Similarly, another taxon from Pakistan was misidentified as A. goossensiae (MCR59; LAH5972011) retrieved from the Genbank that is revised as A. sp. in this study as the ITS sequence is quite different from the type of A. goossensiae.This species is, in fact, closely related to an undescribed species (ZRL2011039) from China.The same species from Pakistan A. sp.(MCR59) clustered close to A. violaceopunctatus, another new taxon reported in this study.
All of the four newly described species in this section were collected from subtropical semiarid and arid regions of Pakistan.Agaricus dunensis was collected from Cholistan desert (L2, L99 and LS4) and forth collection (TS56) from Toba Tek Singh, from sandy soils having a dry arid climate.All the collections of A. glabriusculus were collected from Toba Tek Singh district having subtropical and arid climate.Agaricus badiosquamulosus, is a common species found abundantly in different areas of Lahore and one collection was made from Toba Tek Singh which are subtropical semi-arid and arid areas, respectively.Agaricus robustulus and A. violaceopunctatus, were collected from subtropical region of Lahore.
Mainly, the species of A. sect.Minores have been reported from China (Yunnan) and Thailand.From Thailand, the collections were made from tropical areas while the climate of Yunnan is subtropical.Previously, 27 species were reported from Thailand which are distributed in six clades and 14 species from Yunnan while only three species are shared by both of these regions [7].After the detailed study on A. sect.Minores by Chen et al. 2017 [7], the section comprised of 93 species in total.He et al. 2017 [31] added sixteen new taxa in A. sect.Minores, from tropic origins, China.In this study, multigene and molecular dating analyses have been conducted and very interesting conclusions were made regarding the origin of A. sect.Minores.The study showed that this section has a tropical origin with four major routes: 1) ca.9-13 Ma, species migrated to the Tibetan Plateau and Europe from South Asia; 2) ca.22 Ma, species from outside South Asia dispersed to Europe; 3) around ca. 9 Ma, species spreaded to Alaska via North Asia, then West America from South Asia; and 4) species reached south and Oceania from South Asia by three invading occurrences around ca. 9, 12 and 16-18 Ma, respectively.He et al. 2018 [9] reported three new species of A. sect.Minores based on ITS and multigene phylogenetic data.Recently, Hussain and Sher added two new species in this section from Pakistan [17].In the present study, 41 sequences are added of three newly described species including 29 ITS and six of LSU and Tef-1α regions.So, this section is very diverse having great number of species of tropic origin.

Conclusion
The ITS and multigene phylogenetic analyses interpreted three new species A. badiosquamulosus, A. dunensis, and A. violaceopunctatus, a new record A. robustulus belonging to the Agaricus section Minores from subtropical regions of Pakistan.An already reported taxon from Pakistan and India, A. glabriusculus [17,21] Tef-1α sequences are also added in this study which were not provided in the previous studies.All the micro-and macro-morphological data supported the molecular evidence.The species status of Agaricus cf.goossensiae (ADK2751) and A. goossensiae (MCR 59) on GenBank is clarified by obtaining the ITS sequence data from the type specimen of A. goossensiae (GF929) on special request and Agaricus sp. is assigned to both of these species.In this regard, the current investigation is an important contribution to the previously reported data on this largest section Minores of the genus Agaricus.

Fig 1 .
Fig 1. Maximum likelihood tree of species belonging to A. sect.Minores generated from ITS sequence data.Agaricus campestris is used as outgroup.Bootstrap support values are indicated (BS).New species and new record from Pakistan are in bold.T = type specimen.https://doi.org/10.1371/journal.pone.0302222.g001

Fig 2 .
Fig 2. Maximum likelihood tree of species belonging to A. sect.Minores generated from combined LSU, ITS and TEF1 sequence data.Agaricus campestris is used as outgroup.Bootstrap support values are indicated (BS).New species from Pakistan are in bold red.T = type specimen.https://doi.org/10.1371/journal.pone.0302222.g002

Table 1 .
All the representatives of Agaricus sect.Minores sequences of known and unnamed species are mentioned along with the outgroup and other sections samples.
(A) the newly named species since 2000 have their names ajusted to the left in the column (names in black letters for new species and green letters for renamed species) (B) the newly named species since 2000 have their names ajusted to the left in the column (names in black letters for new species with no colored background and with green background for renamed species) (C) species named before 2000 are adjusted to the right of the column and are in red background (D) the unnamed or provisionally named species (cf.or aff.) are on the left and in blue background ---for samples that are not the type but which have more sequence data (some are paratype P but we have not verified all) ---for samples of the same species later reported from distant countries (generally different continents indicted with initial AF, AS, EU, NA, SA, OC, and CA for the Caribbean) https://doi.org/10.1371/journal.pone.0302222.t001